RESUMEN
Cervical cancer is the fourth-leading cause of cancer deaths among women worldwide and most cases occur in developing countries. Detection of high-risk (HR) HPV, the etiologic agent of cervical cancer, is a primary screening method for cervical cancer. However, the current gold standard for HPV detection, real-time PCR, is expensive, time-consuming, and instrumentation-intensive. A rapid, low-cost HPV detection method is needed for cervical cancer screening in low-resource settings. We previously developed a digital loop-mediated isothermal amplification (dLAMP) assay for rapid, quantitative detection of nucleic acids without the need for thermocycling. This assay employs a microfluidic self-digitization chip to automatically digitize a sample into an array of nanoliter wells in a simple assay format. Here we evaluate the dLAMP assay and self-digitization chip for detection of the commonly tested 14 high-risk HPVs in clinical samples. The dLAMP platform provided reliable genotyping and quantitative detection of the 14 high-risk HPVs with high sensitivity, demonstrating its potential for simple, rapid, and low-cost diagnosis of HPV infection.
Asunto(s)
Alphapapillomavirus , Neoplasias del Cuello Uterino , Detección Precoz del Cáncer , Femenino , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido NucleicoRESUMEN
We present a method to determine the concentration of nucleic acids in a sample by partitioning it into droplets with a nonuniform volume distribution. This digital PCR method requires no special equipment for partitioning, unlike other methods that require nearly identical volumes. Droplets are generated by vortexing a sample in an immiscible oil to create an emulsion. PCR is performed, and droplets in the emulsion are imaged. Droplets with one or more copies of a nucleic acid are identified, and the nucleic acid concentration of the sample is determined. Numerical simulations of droplet distributions were used to estimate measurement error and dynamic range and to examine the effects of the total volume of droplets imaged and the shape of the droplet size distribution on measurement accuracy. The ability of the method to resolve 1.5- and 3-fold differences in concentration was assessed by using simulations of statistical power. The method was validated experimentally; droplet shrinkage and fusion during amplification were also assessed experimentally and showed negligible effects on measured concentration.
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Reacción en Cadena de la Polimerasa/métodos , Estadística como Asunto , Emulsiones , Dispositivos Laboratorio en un Chip , Distribución de Poisson , Reacción en Cadena de la Polimerasa/instrumentaciónRESUMEN
The design and fabrication of a self-digitization dielectrophoretic (SD-DEP) chip with simple components for single-cell manipulation and downstream nucleic acid analysis is presented. The device employed the traditional DEP and insulator DEP to create the local electric field that is tailored to approximately the size of single cells, enabling highly efficient single-cell capture. The multistep procedures of cell manipulation, compartmentalization, lysis, and analysis were performed in the integrated microdevice, consuming minimal reagents, minimizing contamination, decreasing lysate dilution, and increasing assay sensitivity. The platform developed here could be a promising and powerful tool in single-cell research for precise medicine.
Asunto(s)
Dispositivos Laboratorio en un Chip , Ácidos Nucleicos/análisis , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Análisis de la Célula Individual/instrumentación , Diseño de Equipo , Humanos , Células K562RESUMEN
RATIONALE: Surface acoustic wave nebulization (SAWN) is an easy to use sample transfer method for rapid mass spectrometric analysis. A new standing wave (SW) SAWN chip, with higher ionization efficiency than our previously reported design, is used for rapid analysis of lipids. METHODS: The crude, yet fast, Caroff protocol was used for lipid A extraction from Francisella novicida. SW-SAWN with a Waters Synapt G2S quadrupole time-of-flight (QTOF) mass spectrometer was used to generate lipid A ions. Quadrupole collision-induced dissociation (Q-CID) of lipid A at varying CID energies was used to approximate the ion trap MSn data required for our hierarchical tandem mass spectrometry (HiTMS) algorithm. Structural hypotheses can be obtained directly from the HiTMS algorithm to identify species-specific lipid A molecules. RESULTS: SW-SAWN successfully generated ions from lipid A extracted from Francisella novicida using the faster Caroff method. In addition, varying collision energies were used to generate tandem mass spectra similar to MS3 and MS4 spectra from an ion trap. The Q-CID spectra are compatible with our HiTMS algorithm and offer an improvement over lipid A tandem mass spectra acquired in an ion trap. CONCLUSIONS: Combining SW-SAWN and Q-CID enabled more structural assignments than previously reported in half the time. The ease of generating spectra by SAWN tandem MS in combination with HiTMS interpretation offers high-throughput lipid A structural analysis and thereby rapid detection of pathogens based on lipid fingerprinting. Copyright © 2016 John Wiley & Sons, Ltd.
RESUMEN
This paper describes a sample digitization method that generates tens of thousands of nanoliter-sized droplets in a high-density array in a matter of minutes. We show that the sample digitization depends on both the geometric design of the microfluidic device and the viscoelastic forces between the aqueous sample and a continuous oil phase. Our design avoids sample loss: Samples are split into tens of thousands of discrete volumes with close to 100% efficiency without the need for any expensive valving or pumping systems. We envision this technology will have broad applications that require simple sample digitization within minutes, such as digital polymerase chain reactions and single-cell studies.
Asunto(s)
Técnicas Analíticas Microfluídicas/instrumentaciónRESUMEN
Hot embossing is a cost-effective and flexible fabrication technology with high replication accuracy for feature sizes as small as 50 nm. Here we develop a reinforced polydimethylsiloxane (PDMS) mold for hot embossing of cyclic olefin polymer (COP) sheets in the fabrication of microfluidic chips and demonstrate the method by fabricating chips for automated sample digitization in digital nucleic acid assays. The PDMS is hardened by adding an investment powder as a dopant and is constrained with an aluminum frame to prevent lateral expansion during hot pressing. The reinforced PDMS mold demonstrated excellent performance in hot embossing (180 °C, 103 kPa, 5 min) for micropatterning COP sheets, with highly reproducible features as small as 10 µm (width of draining channel). In contrast, the microscale features were inconsistent and distorted when omitting either the investment powder or frame from the PDMS mold. COP chips were assembled by thermally bonding patterned and unpatterned COP sheets. We tested the performance of the COP chip for automated sample digitization in a digital LAMP assay used to quantify human papillomavirus-18 (HPV-18) DNA. A mixture of nucleic acid amplification reagents was loaded into the main channel of the chip using a syringe pump, then the solution was spontaneously partitioned into chambers (â¼0.6 nL), which were then isolated by flowing oil through the chip. The digital LAMP assay produced accurately absolute quantitation of DNA at concentrations ranging from 10 to 1000 copies per µL. The strategy presented here provides a simple, low-cost method to prepare molds for hot embossing, which facilitates rapid validation of microfluidic designs.
Asunto(s)
Cicloparafinas , Ácidos Nucleicos , Humanos , Microfluídica/métodos , Polímeros , Polvos , DimetilpolisiloxanosRESUMEN
We recently reported a new UV-curable polyurethane-methacrylate (PUMA) resin that has excellent qualities as a disposable microfluidic substrate for clinical diagnostic applications. This article discusses strategies to improve the production yield of PUMA chips that contain dense and high-aspect-ratio features, which presents unique challenges in demolding and bonding steps. These fabrication improvements were deployed to produce a microfiltration device that contained closely spaced and high-aspect-ratio columns, suitable for retaining and concentrating cells or beads from a highly diluted suspension.
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Metacrilatos/química , Técnicas Analíticas Microfluídicas/instrumentación , Filtros Microporos , Poliuretanos/química , Neoplasias de la Mama/diagnóstico , Fraccionamiento Celular/instrumentación , Línea Celular Tumoral , Diseño de Equipo , Femenino , HumanosRESUMEN
As microfluidic systems transition from research tools to disposable clinical-diagnostic devices, new substrate materials are needed to meet both the regulatory requirement as well as the economics of disposable devices. This paper introduces a UV-curable polyurethane-methacrylate (PUMA) substrate that has been qualified for medical use and meets all of the challenges of manufacturing microfluidic devices. PUMA is optically transparent, biocompatible, and exhibits high electroosmotic mobility without surface modification. We report two production processes that are compatible with the existing methods of rapid prototyping and present characterizations of the resultant PUMA microfluidic devices.
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Comercio/instrumentación , Diseño de Equipo/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Microfluídica/instrumentación , Farmacopeas como Asunto/normas , Materiales Biocompatibles/química , Metacrilatos/química , Microscopía Electrónica de Rastreo , Poliuretanos/química , Espectrofotometría Ultravioleta , Especificidad por Sustrato , Propiedades de Superficie , Estados UnidosRESUMEN
Rapid food product analysis is of great interest for quality control and assurance during the production process. Conventional quality control protocols require time and labor intensive sample preparation for analysis by state-of-the-art analytical methods. To reduce overall cost and facilitate rapid qualitative assessments, food products need to be tested with minimal sample preparation. We present a novel and simple method for assessing food product compositions by mass spectrometry using a novel surface acoustic wave nebulization method. This method provides significant advantages over conventional methods requiring no pumps, capillaries, or additional chemicals to enhance ionization for mass spectrometric analysis. In addition, the surface acoustic wave nebulization - mass spectrometry method is ideal for rapid analysis and to investigate certain compounds by using the mass spectra as a type of species-specific fingerprint analysis. We present for the first time surface acoustic wave nebulization generated mass spectra of a variety of fermented food products from a small selection of vinegars, wines, and beers.
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Recent single-cell and single-molecule studies have shown that a variety of subpopulations exist within biological systems, such as synaptic vesicles, that have previously been overlooked in common bulk studies. By isolating and enriching these various subpopulations, detailed analysis with a variety of analytical techniques can be done to further understand the role that various subpopulations play in cellular dynamics and how alterations to these subpopulations affect the overall function of the biological system. Previous sorters lack the sensitivity, sorting speed, and efficiency to isolate synaptic vesicles and other nanoscale systems. This paper describes the development of a fluorescence-activated nanoscale subcellular sorter that can sort nearly 10 million objects per hour with single-molecule sensitivity. Utilizing a near-nanoscale channel system, we were able to achieve upward of 91% recovery of desired objects with a 99.7% purity.
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Colorantes Fluorescentes/química , Nanotecnología , Vesículas Sinápticas/química , Animales , Dimetilpolisiloxanos/química , Hidrazinas/química , Técnicas Analíticas Microfluídicas , Microscopía Confocal , Ratas , Vesículas Sinápticas/metabolismoRESUMEN
Quality control is an important and integral part to any microfabrication process. While the widths of features often can be easily assessed by light microscopy, the heights of the fabricated structures are more difficult to determine. Here, we present a rapid, accurate, and low-cost method to measure the heights of microfabricated structures during and after the fabrication process. This technique is based on white-light interferometry, which offers accuracy on the submicrometre scale.
RESUMEN
Over the temperature range 250-300 degrees C, 8-exo-methoxybicyclo[4.2.0]oct-2-ene (1a) undergoes a [1,3] sigmatropic rearrangement to 5-exo- and 5-endo-methoxybicyclo[2.2.2]oct-2-enes, 2a and 2b, respectively, with a clear preference for the si product: si/sr = 3.2. Both 1a and its 8-endo epimer 1b experience appreciable epimerization and fragmentation. A long-lived intermediate with weakly interacting diradical centers, one of which is stabilized by a methoxy substituent, can account for all such observations.